{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T05:52:36Z","timestamp":1781848356124,"version":"3.54.5"},"reference-count":62,"publisher":"Wiley","issue":"1","license":[{"start":{"date-parts":[[2026,4,25]],"date-time":"2026-04-25T00:00:00Z","timestamp":1777075200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"},{"start":{"date-parts":[[2026,4,25]],"date-time":"2026-04-25T00:00:00Z","timestamp":1777075200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/doi.wiley.com\/10.1002\/tdm_license_1.1"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Journal of Microscopy"],"published-print":{"date-parts":[[2026,7]]},"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Since the invention of the electron microscope, morphologists have developed numerous techniques to visualise the native ultrastructure of biological cells and tissues. Although conventional chemical fixation followed by room temperature dehydration remains widely used, it often fails to preserve native structures, leading to distortion and leakage of cellular components. In contrast, cryofixation combined with freeze substitution offers a superior alternative, producing micrographs that more accurately reflect native ultrastructure.<\/jats:p>\n                  <jats:p>We have developed a \u2018sandwich freezing method\u2019 for rapid cryofixation, in which small biological specimens are placed between two copper disks, rapidly frozen by plunging into melting propane cooled with liquid nitrogen, then freeze\u2010substituted in acetone containing osmium tetroxide at low temperature, and finally embedded in epoxy resin.<\/jats:p>\n                  <jats:p>This technique yields exquisitely preserved, near\u2010native ultrastructure in living microorganisms and plant tissues. For cultured cells and animal tissues, prefixation with glutaraldehyde prior to sandwich freezing results in even more native\u2010like preservation compared to specimens frozen without fixation. We propose that glutaraldehyde prefixation prevents the immediate distortion and ultrastructural changes that occur upon removal of cells and tissues from their culture medium or host organism. Importantly, glutaraldehyde rapidly immobilises cellular components without altering cellular architecture.<\/jats:p>\n                  <jats:p>We recommend the routine application of the sandwich freezing\u2013freeze substitution technique to achieve ultrastructural preservation that is as close to the native state as possible in electron microscopy.<\/jats:p>","DOI":"10.1111\/jmi.70096","type":"journal-article","created":{"date-parts":[[2026,4,25]],"date-time":"2026-04-25T11:19:33Z","timestamp":1777115973000},"page":"21-36","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Closer to the native state: Sandwich freezing and freeze\u2010substitution of microorganisms, cultured cells, and animal and plant tissues for electron microscopy"],"prefix":"10.1111","volume":"303","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-1903-1555","authenticated-orcid":false,"given":"Masashi","family":"Yamaguchi","sequence":"first","affiliation":[{"name":"Medical Mycology Research Center Chiba University  Chuo\u2010ku Chiba Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katsuyuki","family":"Uematsu","sequence":"additional","affiliation":[{"name":"Marine Works Japan, Ltd.  Yokosuka Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Toshikatsu","family":"Kuramoto","sequence":"additional","affiliation":[{"name":"Marine Works Japan, Ltd.  Yokosuka Kanagawa Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinichi","family":"Ohno","sequence":"additional","affiliation":[{"name":"University\u00a0of Yamanashi  Chuo\u2010city Yamanashi Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroji","family":"Chibana","sequence":"additional","affiliation":[{"name":"Medical Mycology Research Center Chiba University  Chuo\u2010ku Chiba Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-6652-4654","authenticated-orcid":false,"given":"Azusa","family":"Takahashi\u2010Nakaguchi","sequence":"additional","affiliation":[{"name":"Medical Mycology Research Center Chiba University  Chuo\u2010ku Chiba Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,4,25]]},"reference":[{"key":"e_1_2_19_2_1","doi-asserted-by":"publisher","DOI":"10.3791\/62431"},{"key":"e_1_2_19_3_1","doi-asserted-by":"publisher","DOI":"10.1080\/01913120600932735"},{"key":"e_1_2_19_4_1","doi-asserted-by":"publisher","DOI":"10.1385\/1-59259-201-5:77"},{"key":"e_1_2_19_5_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-59745-294-6_8"},{"key":"e_1_2_19_6_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-3-642-72815-0_8"},{"key":"e_1_2_19_7_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2818.2001.00919.x"},{"key":"e_1_2_19_8_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00418-008-0500-1"},{"key":"e_1_2_19_9_1","first-page":"151","volume-title":"Methods in Cell Biology","author":"Vanhecke D.","year":"2008"},{"issue":"4","key":"e_1_2_19_10_1","first-page":"1295","article-title":"Optimization and application of jet\u2010freezing","volume":"7","author":"M\u00fcller T.","year":"1993","journal-title":"Scanning Microscopy"},{"key":"e_1_2_19_11_1","doi-asserted-by":"publisher","DOI":"10.1111\/1523-1747.ep12371795"},{"key":"e_1_2_19_12_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1365-2818.1976.tb02405.x"},{"key":"e_1_2_19_13_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.81.2.275"},{"key":"e_1_2_19_14_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.91.2.399"},{"key":"e_1_2_19_15_1","doi-asserted-by":"publisher","DOI":"10.1007\/BF00199513"},{"key":"e_1_2_19_16_1","first-page":"265","article-title":"Application of in vivo cryotechnique to the examination of cells and tissues in living animal organs","volume":"21","author":"Terada N.","year":"2006","journal-title":"Histology and Histopathology"},{"key":"e_1_2_19_17_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.87.1.55"},{"key":"e_1_2_19_18_1","doi-asserted-by":"publisher","DOI":"10.1242\/jcs.80.1.253"},{"key":"e_1_2_19_19_1","doi-asserted-by":"publisher","DOI":"10.1038\/308032a0"},{"key":"e_1_2_19_20_1","doi-asserted-by":"publisher","DOI":"10.1017\/S0033583500004297"},{"key":"e_1_2_19_21_1","first-page":"337","article-title":"Cryo\u2010electron microscopy of hepatitis B virus core particles produced by transformed yeast: Comparison with negative staining and ultrathin sectioning","volume":"37","author":"Yamaguchi M.","year":"1988","journal-title":"Journal of Electron Microscopy"},{"key":"e_1_2_19_22_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jsb.2008.01.009"},{"key":"e_1_2_19_23_1","doi-asserted-by":"publisher","DOI":"10.1002\/jemt.1060050305"},{"key":"e_1_2_19_24_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/dfp013"},{"key":"e_1_2_19_25_1","unstructured":"Yamaguchi M. &Yamada H.(2022).Ultrathin sections ofE. coli. In Electron Microscopy Technology Certification Committee (Ed.) Frontispiece 10. New introductory guidebook to electron microscopy (3rd edn.)(in Japanese).International Academic Publishing Co. Ltd."},{"key":"e_1_2_19_26_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/dfx019"},{"key":"e_1_2_19_27_1","doi-asserted-by":"publisher","DOI":"10.1007\/s00709\u2010013\u20100575\u2010y"},{"key":"e_1_2_19_28_1","doi-asserted-by":"publisher","DOI":"10.1007\/978-1-62703-056-4_28"},{"key":"e_1_2_19_29_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/dfaa049"},{"key":"e_1_2_19_30_1","doi-asserted-by":"publisher","DOI":"10.5685\/plmorphol.17.57"},{"key":"e_1_2_19_31_1","first-page":"7","article-title":"A support film of plasma\u2010polymerized naphthalene for electron microscopy: Method of preparation and application","volume":"41","author":"Yamaguchi M.","year":"1992","journal-title":"Journal of Electron Microscopy"},{"key":"e_1_2_19_32_1","doi-asserted-by":"publisher","DOI":"10.1508\/cytologia.87.149"},{"key":"e_1_2_19_33_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/dfae032"},{"key":"e_1_2_19_34_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/dfr032"},{"key":"e_1_2_19_35_1","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.91.3.695"},{"key":"e_1_2_19_36_1","doi-asserted-by":"publisher","DOI":"10.1508\/cytologia.85.15"},{"key":"e_1_2_19_37_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/dfac031"},{"key":"e_1_2_19_38_1","doi-asserted-by":"publisher","DOI":"10.1006\/excr.2002.5578"},{"key":"e_1_2_19_39_1","doi-asserted-by":"publisher","DOI":"10.1078\/0171-9335-00339"},{"key":"e_1_2_19_40_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1574-6968.2009.01643.x"},{"key":"e_1_2_19_41_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/dfp054"},{"key":"e_1_2_19_42_1","doi-asserted-by":"publisher","DOI":"10.3314\/jjmm.46.187"},{"key":"e_1_2_19_43_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2818.2003.01238.x"},{"key":"e_1_2_19_44_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2818.1996.820642.x"},{"key":"e_1_2_19_45_1","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2818.2003.01226.x"},{"key":"e_1_2_19_46_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/52.2.133"},{"key":"e_1_2_19_47_1","first-page":"1","article-title":"Structome of Exophiala yeast cells determined by freeze\u2010substitution and serial ultrathin sectioning electron microscopy","volume":"2","author":"Yamaguchi M.","year":"2006","journal-title":"Current Trends in Microbiology"},{"key":"e_1_2_19_48_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/dfp050"},{"key":"e_1_2_19_49_1","first-page":"138","article-title":"Electron microscopy of hepatitis B virus core antigen expressing yeast cells by freeze\u2010substitution fixation","volume":"47","author":"Yamaguchi M.","year":"1988","journal-title":"European Journal of Cell Biology"},{"key":"e_1_2_19_50_1","first-page":"386","article-title":"Dynamics of hepatitis B virus core antigen in a transformed yeast cell: Analysis with an inducible system","volume":"43","author":"Yamaguchi M.","year":"1994","journal-title":"Journal of Electron Microscopy"},{"key":"e_1_2_19_51_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/51.1.21"},{"key":"e_1_2_19_52_1","doi-asserted-by":"publisher","DOI":"10.1016\/S0378-1097(02)01181-3"},{"key":"e_1_2_19_53_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/dfr052"},{"key":"e_1_2_19_54_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jiac.2024.08.001"},{"key":"e_1_2_19_55_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mimet.2009.09.022"},{"key":"e_1_2_19_56_1","doi-asserted-by":"publisher","DOI":"10.1371\/journal.pone.0117109"},{"key":"e_1_2_19_57_1","doi-asserted-by":"publisher","DOI":"10.3791\/56235"},{"key":"e_1_2_19_58_1","first-page":"29","article-title":"Deep\u2010sea microorganisms and the origin of the eukaryotic cell","volume":"47","author":"Yamaguchi M.","year":"2014","journal-title":"Japanese Journal of Protozoology"},{"key":"e_1_2_19_59_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/dfs062"},{"key":"e_1_2_19_60_1","doi-asserted-by":"publisher","DOI":"10.1093\/jmicro\/dfw016"},{"key":"e_1_2_19_61_1","doi-asserted-by":"publisher","DOI":"10.1508\/cytologia.83.337"},{"key":"e_1_2_19_62_1","doi-asserted-by":"publisher","DOI":"10.1508\/cytologia.85.209"},{"key":"e_1_2_19_63_1","first-page":"209","article-title":"Convenient method for better preservation of fine structures of cultured macrophages and engulfed yeast cells by freeze\u2010substitution fixation","volume":"66","author":"Yamaguchi M.","year":"2017","journal-title":"Microscopy"}],"container-title":["Journal of Microscopy"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/jmi.70096","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/full-xml\/10.1111\/jmi.70096","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/pdf\/10.1111\/jmi.70096","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,6,19]],"date-time":"2026-06-19T05:23:52Z","timestamp":1781846632000},"score":1,"resource":{"primary":{"URL":"https:\/\/onlinelibrary.wiley.com\/doi\/10.1111\/jmi.70096"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,4,25]]},"references-count":62,"journal-issue":{"issue":"1","published-print":{"date-parts":[[2026,7]]}},"alternative-id":["10.1111\/jmi.70096"],"URL":"https:\/\/doi.org\/10.1111\/jmi.70096","archive":["Portico"],"relation":{},"ISSN":["0022-2720","1365-2818"],"issn-type":[{"value":"0022-2720","type":"print"},{"value":"1365-2818","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,4,25]]},"assertion":[{"value":"2025-10-07","order":0,"name":"received","label":"Received","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-04-10","order":2,"name":"accepted","label":"Accepted","group":{"name":"publication_history","label":"Publication History"}},{"value":"2026-04-25","order":3,"name":"published","label":"Published","group":{"name":"publication_history","label":"Publication History"}}]}}